Recently, a need for checking the amount of glucose in blood (blood glucose) periodically in diagnosing and preventing diabetes has been increased. Such blood glucose measurement may be carried out readily using a palm portable glucose meter and, more particularly, using a biosensor of test strip form for measuring blood glucose individually. However, since the blood glucose varies considerably according to the diet status and body status of a patient and the process of extracting blood necessary for the measurement is accompanied with pain, it is difficult for the patient to measure the blood glucose in conformity with its prescriptions exactly.
Glycated proteins, particularly, glycated hemoglobins are modified proteins produced through an Amadori rearrangement by reacting upon the glucose in blood for many hours. It has been well known that such glycated proteins are closely associated with an average level of blood glucose of a diabetic for two to three months. Accordingly, ordinary measurements of the blood glucose are important for the diabetic to confirm the daily health status and take necessary steps and, furthermore, the measurement of glycated proteins/hemoglobins is necessary to analyze the long term blood glucose and make an appropriate treatment, if necessary.
Standard measurement of glycated proteins is carried out in such a manner that the blood sample collected is mixed with an appropriate sample, e.g., a surfactant such as triton x-100, to be hemolyzed; the resulting sample is passed through a high performance liquid chromatography charged with boronic acid derivatives to be divided from normal proteins; and specific wavelengths of the proteins divided by visible rays are measured, thus calculating the glycation ratio. However, such standard measurement should be carried out by an expert using expensive chromatography equipment in a clinical laboratory well established. Accordingly, various researches aimed at developing method and apparatus for detecting the amount of glycated proteins readily to grasp the exact status of a diabetic in the field have continued to progress.
U.S. Pat. No. 5,541,117 has disclosed a method for determining glycated hemoglobin in blood comprising: treating a blood sample which contains the glycated hemoglobin to be hemolyzed; contacting the hemolyzed blood sample with a first immune reactant that specifically binds to glycated hemoglobin and a second immune reactant which binds to the first immune reactant; and determining binding of the first immune reactant to the second immune reactant as a determination of glycated hemoglobin in the blood sample. U.S. Pat. No. 6,677,158 has disclosed a method, similar to the former, for measurement of glycated hemoglobin by a rapid strip test procedure, in which boronic acid derivatives as a compound capable of coupling with glycated proteins selectively, instead of the immune reactants, are charged into a porous pad. Such immune chromatography method using the rapid test procedure can determine the ratio of glycated hemoglobin with ease, whereas, it has some drawbacks in that it should use expensive antibodies; it is difficult to manufacture the products having a regular quality due to the non-uniformity of the porous pad itself, used as a developing sheet; and its result is semi-quantitative.
Meanwhile, U.S. Pat. No. 5,242,842 has disclosed a method of assessing glycosylated hemoglobin in a sample containing both glycosylated and non-glycosylated hemoglobin in solution, in which the sample solution is contacted with signal-forming molecules to form a reaction mixture containing glycosylated hemoglobin having the signal-forming molecule; glycosylated hemoglobin having the signal-forming molecule is precipitated from the reaction mixture by a non-immobilized precipitating agent; and the precipitate is separated from the reaction mixture, thus assessing the signal-forming molecules which are bound to the separated hemoglobin and assessing the separated glycosylated and non-glycosylated hemoglobin. Such method provides an easy analysis using simplified portable equipment since the concentration of the signal-forming molecules bound to or separated with the hemoglobins may be determined from the strength of the signal obtained from the signal-forming molecules. U.S. Pat. No. 5,631,364 has disclosed labeled boronic acid derivatives useful for analyzing glycated proteins, in which an additional process for washing boronic acid derivatives, not bound to glycated proteins, is required and desired results can be obtained only when the exact amount of sample is applied to.
European Patent No. EP0194084 has disclosed a method an electrochemical assay for electrochemically assaying the mount of glycosylated proteins using ferrocene boronic acid or its derivative as a mediator for a wide range of oxido-reductases. However, such method cannot be utilized practically, since it cannot completely exclude the influence of glucose contained in blood. U.S. Pat. No. 6,054,039 has disclosed a method of determining the concentration of glycoproteins and glycosylated hemoglobin, in which porous films impregnated with compounds that cause oxidation-reduction reactions with glycoproteins are laminated to a sensing electrode having a mediator and a reference electrode is arranged faced with the sensing electrode. However, according to the method, the arrangement of the electrode sensors is complicated and it is difficult to estimate the relative amount of glycoproteins.
In addition, several methods applicable to the portable measurement instrument of glycated proteins have been disclosed in European Patent No. EP0455225, U.S. Pat. Nos. 6,174,734 and 6,162,645. Such methods are for determining the relative amount of glycated proteins by using enzyme-antibody conjugates or boronic acid derivative labeling compounds after separating proteins in a sample using a solid phase to which immune antibodies that couple with both glycated proteins and non-glycated proteins are immobilized. Furthermore, U.S. patent application Ser. No. 20030073243 has disclosed a method for quantitative determination of glycated hemoglobin, in which glycated hemoglobins are separated from non-glycated hemoglobins to determine the amount of glycated hemoglobins coupled with a solid phase surface via coloring method using catalytic properties of glycated hemoglobins for hydrogen peroxide.
The related arts cited above are all directed to a method of measuring glycated proteins spectroscopically or electrochemically by coupling markers with glycated proteins.
Accordingly, the inventors of the present invention have learned that it is possible to determine the amount of glycated proteins by measuring the amount of remaining markers separated after coupling with glycated proteins and completed the present invention.